The GM2 Activator Protein (GM2AP) plays an essential role in the catabolism of GM2 in lysosomes. GM2 is a complex glycosphingolipid (GSL) that is a degradation intermediate in the breakdown of GM1 which is found in relatively high concentrations in the outer surface of plasma membranes in neuronal cells. GM2AP acts as a substrate specific co-factor by solubilizing GM2 from the intralysozomal vesicles and presenting the oligosaccharide moiety to beta-hexosaminidase A (HexA) for hydrolytic cleavage. Mutations in either HexA or GM2AP lead to the storage of GM2 within lysozomes and cell death. A well known example of this type of lysosomal storage disease is Tay-Sachs syndrome. The mechanism for how the GM2AP accessory protein regulates enzymatic degradation of GM2 as the first step in the GM2 lipid metabolism remains unclear. The proposed work will focus on elucidating the molecular details of this mechanism through site-directed spin labeling anomposition of late endosomes and intralysosomal vesicles are believed to alter the bilayer physical properties allowing GM2AP to extract GM2 from membranes. The current hypothesis is that in membranes of high curvature of neuronal lysozome composition, the binding is a transient event. Fluorescence based binding measurements will be used to test this hypothesis. Recently, crystal structures of the GM2AP bound to GM2, POPG and PAF were reported. The model of conformational changes predicted by X-ray crystallography data for specific and non-specific substrates will be tested by SDSL EPR experiments of GM2AP in solution where GM2 is extracted from micelles, as well as from lipid bilayers. Currently, the GM2AP has been expressed in E.coli, insect cells and in yeast. The effects of glycosylation on the membrane binding properties will also be examined. The general relevance of this research is that congenital mutations in GM2AP or HexA result in lysozomal storage diseases. The GM2-gangliosidoses (including Tay-Sachs disease) are a group of inherited disorders that result from defects in the catabolism of GM2. In fact, a variety of lysosomal storage diseases (Pompe, Fabry and the gangliosidoses) result from aberrations in lipid metabolism. A more detailed understanding of this critical first step in GM2 catabolism may lead to developments in drug or gene therapies for these fatal diseases. [unreadable] [unreadable] [unreadable] [unreadable]